MELATONIN SENSITIZES HUMAN BREAST CANCER CELLS TO IONIZING RADIATION
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1 DEPARTMENT OF PHYSIOLOGY AND PHARMACOLOGY UNIVERSITY OF CANTABRIA, SANTANDER, SPAIN MELATONIN SENSITIZES HUMAN BREAST CANCER CELLS TO IONIZING RADIATION Crolin Alonso-González
2 ANDROGENS ESTROGENS PERIPHERAL TISSUES Adipose tissue, skin, osteoblsts, condrocites, vsculr endothelium, ortic soft muscle, brin (hypothlmus, tonsil) ESTROGENS ANDROGENS ADRENAL Inhibition of estrogen ctions SERMs ANTIESTROGENS x MAMMARY TUMOR ANDROGENS MELATONIN EPITHELIAL CELLS FIBROBLASTS ADIPOCYTES Inhibition of estrogen synthesis SEEMs AROMATASE INHIBITORS x ESTROGENS Biologiclly inctive estrogens
3 IRRADIATION (dose rnging 4-12 Gy) Cell prolifertion Meltonin 1 mm Meltonin 1 µm Meltonin 1 nm 7 Dys 3-6 dys Cell cycle phse distribution RAD51 nd DNA-PKs gene expression Mlignnt epithelil cells MCF-7
4 Cell prolifertion (% of control) Cell prolifertion (% of control) EFFECTS OF MELATONIN ON BREAST CANCER CELL PROLIFERATION b c 5 1 µm 1 mm 1 nm Control 1 mm 1 µm Meltonin 1 nm 3 6 Dys, p<.1 vs Control b, p<.1 vs Meltonin 1 mm c, p<.1 vs Meltonin 1 µm Alonso-González et l., J Pinel Res 58: , 215.
5 % of cell prolifertion with respect to control non rdited cells EFFECTS OF IONIZING RADIATION ON BREAST CANCER CELL PROLIFERATION (MCF-7) 12 Gy Dys 4 Gy 8 Gy 12 Gy Alonso-González et l., J Pinel Res 58: , 215
6 Cell Prolifertion (% of control rdited cells) POTENTIATION OF RADIATION-INDUCED GROWTH INHIBITION BY MELATONIN 1 b c b c 5, p<.1 vs Control b, p<.1 vs Meltonin 1 mm c, p<.1 vs Meltonin 1 µm Before irrdition Before nd fter irrdition Before irrdition Before nd fter irrdition Before irrdition Before nd fter irrdition Control Meltonin 1 mm Meltonin 1 µm Meltonin 1 nm Alonso-González et l., J Pinel Res 58: , 215
7 CELL CYCLE DISTRIBUTION IN MCF-7 CELLS AFTER TREATMENT WITH MELATONIN AND IONIZING RADIATION Cell cycle distribution Cell cycle distribution Cell cycle distribution Cell cycle distribution 1 Non-rdited cells 1 Meltonin pretreted cells 5 5 G 1 S G 2 -M G 1 S G 2 -M 1 Rdited cells 1 Meltonin Pretreted nd Rdited cells 5 G 1 S G 2 -M, p<.1 vs Non-rdited cells b, p<.1 vs Rdited cells 5 b b b G 1 S G 2 -M Alonso-González et l., J Pinel Res 58: , 215
8 Speidel, Arch Toxicol 89:51-517, 215
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10 IR DNA DAMAGE DOUBLE STRAND BREAKS (DSBs) ATM MRN COMPLEX P53 CHK2 Apoptosis Senescence P53 CHK2 Cell cycle rrest DNA dmge repir Genomic Integrity Restored SURVIVAL
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12 REPAIR OF DNA DOUBLE STRAND BREAKS (DSBs) NHEJ HR M G 2 G 1 NHEJ S HR
13 RAD51 (% of control non-rdited cells) RAD51 (% of control rdited cells) EFFECTS OF RADIATION AND MELATONIN ON RAD51 mrna EXPRESSION IN MCF-7 CELLS 15 1 p< b c b d e 5 Control 1 mm 1 µm 1 nm Non-Rdited cells Rdited cells Meltonin pretretment before rdition, p<.1 vs Control b, p<.1 vs Meltonin 1 mm c, p<.1 vs Meltonin 1 µm RADIATION Alonso-González et l., J Pinel Res 58: , 215
14 DNA-PKcs (% of control non-rdited cells) DNA-PKcs (% of control rdited cells) EFFECTS OF RADIATION AND MELATONIN ON DNA-PKcs mrna EXPRESSION IN MCF-7 CELLS 15 1 p< b c 5 Control 1 mm 1 µm 1 nm Non-Rdited cells Rdited cells, p<.1 vs Control b, p<.1 vs Control c, p<.1 vs Meltonin 1 mm d, p<.1 vs Meltonin 1 mm e, p<.1 vs Meltonin 1 µm Meltonin pretretment before rdition RADIATION Alonso-González et l., J Pinel Res 58: , 215
15 Gtz nd Wiesmüller, Cell Deth Differ 13:3-116, 26
16 (% of control non-rdited) EFFECTS OF IONIZING RADIATION AND MELATONIN ON p53 mrna EXPRESSION IN BREAST CANCER CELLS b c d 2 b 15 5 Control Non- Rdited cells Control rdited cells 1 mm 1 µm 1 nm Meltonin pretretment before rdition, p<.1 vs Control non-rdited cells b, p<.1 vs Control rdited cells c, p<.1 vs Meltonin 1 mm d, p<.1 vs Meltonin 1 µm RADIATION
17 Decresing cell prolifertion. MELATONIN PRETREATMENT BEFORE RADIATION SENSITIZES BREAST CANCER CELLS TO THE IONIZING EFFECTS OF RADIATION p53 Inducing cell cycle rrest. Down-regulting RAD-51 nd lso DNA-PKc. Two proteins involved in doublestrnd DNA brek repir mechnisms.
18 RESEARCH GROUP CAROLINA ALONSO-GONZÁLEZ ALICIA GONZÁLEZ CABEZA CARLOS MARTÍNEZ CAMPA JAVIER MENÉNDEZ MENÉNDEZ SAMUEL COS CORRAL Technicl ssistnce José Antonio de Cos Cossío Gem Vir Ruíz
19 DHEA ESTRONE ANDROSTENEDIONE SULFATE DHEA-S blood ESTRADIOL SULFATE Estrone sulftse DHEA 17ß-Hydroxysteroid dehydrogense Estrdiol sulftse ANDROSTENEDIOL ANDROSTENEDIONE P45 Aromtse Estrone sulfotrnsferse ESTRONE 17ß-Hydroxysteroid dehydrogense 17ß-Hydroxysteroid dehydrogense 17β-HSD type 1 Oxidtive forms Reductnt forms TESTOSTERONE ESTRADIOL P45 Aromtse Estrdiol sulfotrnsferse Brest crcinom epithelil cell
20 (% of control non-rdited cells) (% of control non-rdited cells) EFFECTS OF IONIZING RADIATION AND MELATONIN ON AROMATASE ACTIVITY AND mrna EXPRESSION IN HUMAN BREAST CANCER CELLS AROMATASE ACTIVITY AROMATASE mrna EXPRESSION b c b 5 b c b Control Control Non- rdited Rdited cells cells 1 mm 1 µm 1 nm Meltonin pretretment before rdition Control Control Non- rdited Rdited cells cells 1 mm 1 µm 1 nm Meltonin pretretment before rdition Rdition, p<.1 vs Control non-rdited cells b, p<.1 vs Control rdited cells c, p<.1 vs Meltonin 1 mm Rdition
21 (% of control non-rdited) (% of control non-rdited) EFFECTS OF IONIZING RADIATION AND MELATONIN ON THE TWO MAJOR PROMOTERS (pi.3 AND pii) DRIVING AROMATASE EXPRESSION IN BREAST CANCER Promoter I.3 Promoter II b d c d e 5 b d b d Control Control Non- rdited Rdited cells cells 1 mm 1 µm 1 nm Meltonin pretretment before rdition Control Control Non- rdited Rdited cells cells 1 mm 1 µm 1 nm Meltonin pretretment before rdition Rdition Rdition, p<.1 vs Control non-rdited cells b, p<.1 vs Control rdited cells c, p<.1 vs Control rdited cells d, p<.1 vs Meltonin 1 mm e, p<.1 vs Meltonin 1 µm
22 (% of control non-rdited) EFFECTS OF IONIZING RADIATION AND MELATONIN ON AROMATASE PROMOTER I.7 mrna EXPRESSION IN BREAST CANCER CELLS 15 1 Promoter I.7 c b d c e 5 Control Control Non- rdited Rdited cells cells 1 mm 1 µm 1 nm Meltonin pretretment before rdition, p<.1 vs Control non-rdited cells b, p<.1 vs Control rdited cells c, p<.1 vs Control rdited cells d, p<.1 vs Meltonin 1 mm e, p<.1 vs Meltonin 1 µm Rdition
23 (% of control non-rdited cells) (% of control non-rdited cells) EFFECTS OF IONIZING RADIATION AND MELATONIN ON SULFATASE ACTIVITY AND mrna EXPRESSION IN HUMAN BREAST CANCER CELLS SULFATASE ACTIVITY SULFATASE mrna EXPRESSION b b c b d 5 b b c b c d Control Control Non- rdited Rdited cells cells 1 mm 1 µm 1 nm Meltonin pretretment before rdition Control Control Non- rdited Rdited cells cells 1 mm 1 µm 1 nm Meltonin pretretment before rdition Rdition, p<.1 vs Non-rdited cells b, p<.1 vs Control rdited cells c, p<.1 vs Meltonin 1 mm d, p<.1 vs Meltonin 1 µm Rdition
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28 CELL CYCLE DISTRIBUTION IN MCF-7 CELLS AFTER TREATMENT WITH MELATONIN AND IONIZING RADIATION h Control Meltonin 1 mm 1 µm 1 nm G 1 51 ±.8 67 ±.6 * 65.3 ± 1.3 * 69 ±.3 * S 28 ± ± 1.2 * 19.9 ±.9 * 18 ±.8 * G 2 /M 21 ± ±.8 * 14.3 ± 1.1 * 13 ±.2 * 6 h fter tretment G 1.8 ±.7 42 ±.6 * 31.2 ± ±.5 S 45.8 ±.5 38 ± ± ±.9 G 2 /M 28.4 ±.6 2 ±.8 *.3 ± ±.4 12 h fter tretment G ± ± ± ±.5 S 7.1 ± ± ± ±.9 G 2 /M 33.6 ±.5 36 ± ± ±.2 24 h fter tretment G ± ±.7 * 56.6 ± ±.4 * S 13.8 ±.5 7 ± 1.2 * 12.9 ± ±.8 * G 2 /M 3.6 ±.1 24 ±.4 * 29.9 ±.2.6 ±.1 *
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30 MELATONIN PRETREATMENT BEFORE RADIATION SENSITIZES BREAST CANCER CELLS TO THE IONIZING EFFECTS OF RADIATION p53 Decresing tumorl estrogen production: inhibition of romtse nd sulftse ctivity nd expression. Decresing cell prolifertion. Inducing cell cycle rrest. Down-regulting RAD-51 nd DNA-PKcs, two proteins involved in double-strnd DNA brek repir.
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